J. Molec. Microbiol. Biotechnol. (1999) 1(1): 93-100. Microbial Adaptations to the Psychrosphere/PiezosphereJMMB Symposium 93

Microbial Adaptations to the Psychrosphere/Piezosphere

review genetic and biochemical studies of pressure Douglas H. Bartlett* adaptation by psychrotolerant/psychrophilic piezophilic Center for Marine Biotechnology and Biomedicine prokaryotes with an emphasis on results obtained from Scripps Institution of Oceanography my lab with Photobacterium profundum. Other reviews University of California, San Diego covering high pressure adaptation by low temperature- La Jolla, CA 92093-0202, USA adapted deep-sea organisms include those written by Bartlett (1992), DeLong (1992), Prieur (1992), Somero (1990; 1991; 1992), Bartlett et al., (1995), Yayanos (1995; 1998), Kato and Bartlett (1997), and within this miniseries Abstract symposium the article by Kato and Qureshi.

Low temperature and high pressure deep-sea Basis of Pressure Effects environments occupy the largest fraction of the biosphere. Nevertheless, the molecular adaptations The thermodynamic consequences of changes in pressure that enable life to exist under these conditions remain are straightforward. Elevated pressure promotes poorly understood. This article will provide an overview decreased system volume changes associated with the of the current picture on high pressure adaptation in equilibria and rates of biochemical processes. Thus, life in cold oceanic environments, with an emphasis on the deep sea must entail life at low volume change, or genetic experiments performed on Photobacterium other biochemical adjustments which compensate for the profundum. Thus far genes which have been found or consequences of elevated pressure. The relationship of implicated as important for pressure-sensing or pressure to equilibrium and rate processes can be pressure-adaptation include genes required for fatty expressed mathematically as follows: acid unsaturation, the membrane protein genes toxR and rseC and the DNA recombination gene recD. Many 1) Kp = K1 exp(-P∆V/RT) deep-sea possess genes for the production ‡ of omega-3 polyunsaturated fatty acids. These could and 2) k p = k1 exp(-P∆V /RT) be of biotechnological significance since these fatty acids reduce the risk of cardiovascular disease and These two equations express the relationship of either the certain cancers and are useful as dietary supplements. equilibrium constant (K) or rate constant (k) of a reaction to pressure, as determined by the size of the volume Introduction change that takes place during either the establishment of equilibrium (∆V) or the formation of the activated complex ‡ Low temperature and high pressure environments (the (∆V ). K1 and k1 are the equilibrium and rate constants, psychrosphere and the piezosphere, respectfully) occupy respectively, at atmospheric pressure; Kp and kp are the the largest fractions of the known biosphere in terms of constants at a higher pressure, R is the gas constant and volume. Within these environments psychrophiles exist T is absolute temperature. Because the equilibrium and which can grow at temperatures as low as -12 °C (Russell, rate constants are related to pressure in a logarithmic 1990), and piezophiles (also termed barophiles) exist which fashion, seemingly small changes in volume can lead to can grow at pressures as high as 130 megapascal large changes in K or k. For example, a volume change of [Yayanos, 1998; 1 megapascal (MPa) = 10 bar ≅ 9.9 +50 cm3mol-1 will lead to a 89% inhibition of a rate (or 89% atmospheres]. The lower temperature limit is apparently change in K value) at 100 MPa pressure. A volume change set by the freezing temperature of intracellular water, of +100 cm3mol-1 will lead to a 35% decrease in k or K at whereas the upper pressure limit for life is not known. 10 MPa, and >99% decrease at 100 MPa. Volume changes Although Archaea, particularly those belonging to the accompanying many biological processes (both equilibria nonthermophilic branch within the Crenarchaeota kingdom, and reaction rates) are in the range of approximately 20- are highly abundant in low temperature and high pressure 100 cm3mol-1. Therefore, pressure changes can lead to environments (Fuhrman et al., 1992; DeLong et al., 1994), very substantial alteration (decrease or increase) of all of the cultured psychrotolerant/psychrophilic piezophilic biological activities and structures in the absence of prokaryotes fall within well known lineages of the Bacterial adaptation. domain. Results to date indicate that the adaptations that Because water itself is not highly compressible (about enable these deep-sea microorganisms to grow under low 4% per 100 MPa pressure), the effects of increased temperature and high pressure conditions entail subtle pressure on biochemical processes are not due to effects variations of certain regulatory processes and aspects of stemming from compression of the aqueous medium. cellular architecture present in mesophiles. Here I will briefly However, water structure is very important in pressure sensitivity because changes in hydration of macromolecules and metabolites can lead to substantial *For correspondence. Email [email protected]; Tel. 619-534-5233; Fax. 619-534-7313. alterations in system volume.

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Order from caister.com/order 94 Bartlett

Changes in hydrostatic pressure perturb the volume Fatty Acid Regulation of a system without changing its internal energy (as occurs with temperature), or its solvent composition (as occurs Among the evolutionarily adaptive and transiently with pH and osmolarity changes). Thus, pressure is both acclimatory changes thought to be important for growth at a unique and a fundamental thermodynamic parameter. either low temperature or high pressure, membrane fatty acid alterations have received the most attention (Russell Identification of Pressure Sensitive and Hamamoto, 1998; Yayanos, 1998). Both decreased Processes in Mesophilic Microorganisms temperature and increased pressure cause fatty acyl chains to pack together more tightly and influence gel state (Lß) Although the basis of pressure effects on many simple to liquid crystalline (Lα) state transitions of membranes chemical processes have been described, pinpointing the (reviewed by Bartlett and Bidle, 1999; Suutari and Laakso, physical nature of pressure effects on biological systems 1994). For many phospholipids and natural membranes is extremely complicated. Changes in pressure, as with the transition slope in a temperature, pressure-phase changes in other physical parameters, can affect a variety diagram for the Lß - Lα main transition is approximately of cellular processes. Experimentally, cell division, flagellar 20 °C/100 MPa at pressures lower than 100 MPa. Based function, and more critically, DNA replication, protein on this relationship the combined effect of pressure and synthesis, enzyme function, and membrane structure have temperature on the phase state of a membrane from a all been found to be sensitive to elevated pressure in deep-sea bacterium existing at 100 MPa and 2 °C is shallow-water bacteria (reviewed in Bartlett, 1992). Many equivalent to an identical membrane at atmospheric of these studies have been undertaken using the pressure and -18 °C (lower than the low temperature limit moderately piezotolerant , where it has for life). Decreased temperature or increased pressure been found that pressures higher than 10 MPa inhibit have been found to result in increased unsaturation, chain motility, 20 -50 MPa; cell division, 50 MPa; DNA replication, length and the ratio of anteiso to iso branching, as well as 58 MPa; protein synthesis, and 77 MPa; RNA synthesis. decreased chain lengths. These changes may represent When Escherichia coli was subjected to 55 MPa the manifestations of “homeoviscous adaptation”, the theory number of polypeptides synthesized was greatly reduced, espoused by Sinensky (1974) whereby organisms adjust but many proteins exhibited elevated rates of synthesis their membrane composition in response to changes in relative to total protein synthesis (Welch et al., 1993). As temperature in order to maintain a nearly constant with other stress responses, this pressure-inducible protein membrane microviscosity. It has also been suggested that (PIP) response was transient. The protein exhibiting the “homeophasic adaptation”, maintaining a certain greatest pressure inducibility was a small basic protein not percentage of membrane phospholipids within a liquid induced by any other known stress conditions. Among the crystalline state, is more physiologically relevant than the PIPs identified in E. coli many present an interesting actual level of membrane fluidity (McElhaney, 1982). paradox. High pressure induces more heat shock proteins Although once thought to be virtually nonexistent in (eleven) than most other conditions outside of those which bacteria, omega-3 polyunsaturated fatty acids (PUFAs) are precisely mimic a heat shock response, while also inducing also frequently present in the lipids of microbes from low more cold shock proteins (four) than most conditions temperature and high pressure environments. There is an outside of those which precisely mimic a cold shock increased proportion of docosahexaenoic acid (DHA; 22:6) response. The high pressure induction of heat shock and/or eicosapentaenoic acid (EPA; 20:5) found in proteins has been observed in many prokaryotic and microbes isolated from deep-sea versus shallow water eukaryotic microorganisms as well as human cells in tissue animals (Yano et al., 1997). Higher percentages of EPA culture (reviewed by Bartlett et al., 1995). containing bacteria have also been noted in Antarctic Heat shock and cold shock proteins have inverse versus temperate bacteria (Nichols et al., 1993). responses to a variety of conditions (VanBogelen and Neidhardt, 1990) so why should high pressure turn on both Photobacterium profundum as a Model System for heat shock and cold shock proteins? Both cold temperature Studying Pressure Adaptation and high pressure inhibit an early step of protein synthesis. In addition, these conditions also both decrease membrane The only deep-sea isolate to yet be employed for genetic fluidity, increase nucleic acid secondary structure and either studies of pressure adaptation is Photobacterium extremes of temperature as well as high pressure can profundum strain SS9. Members of this species have been reduce the stability of multimeric proteins (reviewed in isolated from amphipods in the Sulu Sea recovered from Bartlett et al., 1995) . The high pressure induction of heat 2551 m and from sediment obtained from 5110 m in the shock and cold shock proteins helps to underscore the Ryukyu Trench (DeLong, 1986; Nogi et al., 1998). SS9 unique and pleiotropic consequences of shifts in pressure has a relatively small genome size of 2 Mbp (Smorawinska on cell functions. The relationship between high pressure and Kato; unpublished results), a temperature optimum of and high temperature stress is further extended by the 15 °C and a pressure optimum of 20 - 30 MPa (DeLong, observation that high temperature preincubation of the 1986). Why should this moderately piezophilic yeast Saccharomyces cerevisiae confers significant psychrotolerant species represent a useful model system protection against lethal levels of hydrostatic pressure for studies of life at high pressure and low temperature? It (Komatsu et al., 1991). How Hsps and other heat-inducible has a relatively rapid doubling time of 2 hours under optimal factors protect cells from high pressure stress is as yet growth conditions and grows well over a wide temperature unknown but could involve protecting membrane or protein range (the 4 °C rate is nearly identical to the 15 °C rate) stability. and pressure span (growth at 1 and 30 MPa are nearly identical and the upper pressure limit is 90 MPa). Microbial Adaptations to the Psychrosphere/Piezosphere 95

Furthermore, it is possible to introduce a variety of broad The progress which has been made in recent years host range plasmids into SS9 by conjugal transfer, and to towards understanding the genetic basis of EPA synthesis perform transposon mutagenesis (Tn5 and mini-Mu), has been used for directly testing the role of EPA in low interposon mutagenesis and allelic exchange (Chi and temperature and high pressure growth of strain SS9. Much Bartlett, 1993; 1995; Bartlett and Chi, 1994; Welch and of the driving force for this research has been that omega- Bartlett, 1996; 1998). Finally, SS9 responds to pressure 3 fatty acids such as EPA and DHA are considered shifts by modulating the abundance of a number of fatty important chemicals for biotechnology because of their acids and proteins as described below. As can be seen in activities in reducing the risk of cardiovascular disease and Table 1, many of the genes obtained thus far from certain cancers, their use as dietary supplements in piezophilic bacteria have been isolated from strain SS9. mariculture and poultry farming, and their application as substrates for the synthesis of certain hormone-like Fatty Acid Regulation in P. profundum molecules (Simopoulos, 1991). The molecular biology of PUFA production began when a 38 kbp genomic DNA As with many other deep-sea bacteria, strain SS9 fragment was isolated from a Shewanella putrefaciens enhances the proportion of both monounsaturated species (neither psychrophilic or piezophilic) which (MUFAs) and polyunsaturated fatty acids (PUFAs) when conferred EPA production ability to both E. coli and grown at decreased temperature or elevated pressure. In Synechococcus (Yazawa, 1996; Takeyama et al., 1997). order to assess the importance of unsaturated fatty acids The nucleotide sequence of this DNA was determined and (UFAs) to microbial growth at low temperature and high based upon deletion analysis and deduced amino acid pressure, the effect of treatments and mutations altering homology to other known enzymes involved in fatty acid UFA levels in strain SS9 was investigated (Allen et al., synthesis, heterocyst glycolipid synthesis and polyketide 1999). Treatment of cells with the ß-ketoacyl ACP synthase synthesis, six open reading frames were identified inhibitor cerulenin inhibited MUFA but not saturated fatty as important to EPA production (Yazawa, 1996). This acid or PUFA synthesis, and led to decreased growth rate genetic structure is very similar among diverse EPA and and yield at low temperature and high pressure in the DHA producing bacteria (D. Facciotti, Calgene Inc.; absence of exogenous MUFA. The inspection of UFA personal communication). Because PUFA producing auxotrophic mutants also indicated a role for MUFAs bacteria are present in many separate lineages, it is (particularly cis-vaccenic acid, 18:1) in growth of SS9 at possible that the genes for PUFA biosynthesis have been low temperature and elevated pressure. One of these acquired in many instances via horizontal gene transfer. mutants, strain EA3, was deficient in the production of Figure 1 shows a schematic of the S. putrefaciens SCRC- MUFAs and was sensitive to both low temperature and 2738 genes involved in EPA biosynthesis and the high pressure in the absence of exogenous 18:1 fatty acid. relationship of their gene products to other bacterial Another mutant, strain EA2, produced little MUFAs but proteins. elevated levels of the PUFA species eicosapentaenoic acid Based on the sequence of the SCRC-2738 EPA genes (EPA; 20:5n-3). This mutant grew slowly, but was not low it was possible to employ the polymerase chain reaction temperature-sensitive or high pressure-sensitive. These to amplify a portion of the SS9 EPA biosynthesis gene results have provided the first evidence that UFAs are cluster and to use this DNA fragment to construct an EPA required for low temperature and high pressure adaptation, mutant using reverse genetics (Allen et al., 1999). Much and have also suggested that overproduction of PUFAs to our surprise this mutant displayed no growth defects might at least partially compensate for reduced MUFA whatsoever, including under conditions of low temperature levels. The fact that none of the MUFA mutants displayed and elevated pressure. Two explanations for this lack of reduced EPA levels further indicated that the production of altered phenotype could be that the increased MUFA these two types of UFAs are controlled by separate content of the EPA mutant is capable of compensating for biosynthetic pathways. the absence of EPA or that EPA is only required under

Table 1. Genes Cloned from Deep-Sea Piezotolerant/Piezophilic Bacteria

Gene Protein/function Source organism(s) Isolation Depth (m) Reference asd aspartate ß-D-semialdehyde Shewanella DSS12 5110 and 6356 (Kato, et al., 1997) dehydrogenase and DB6705 cydD, cydC cytochrome bd biosynthesis Assorted Shewanellas up to 11000 (Kato, et al., 1995; Kato, et al., 1996; Kato, et al., 1997; Kato et al., 1997; Kato et al., 1997; Li et al., 1998) mdh malate dehydrogenase P. profundum SS9 2551 (Welch and Bartlett, 1997) ompH, ompL probable porins P. profundum SS9 2551 (Bartlett et al., 1993; Welch and Bartlett, 1996) recD Exonuclease V subunit P. profundum SS9 2551 (Bidle and Bartlett, 1999) rpsD, rpoA, rplQ RNA polymerase a subuni Shewanella DB6705 (Nakasone et al., 1996) t and ribosomal proteins rpoE operon RNA polymerase s subunit P. profundum SS9 2551 (Chi and Bartlett, 1995) and regulatory proteins ssb Single-stranded-DNA- Various Shewanella up to 8600 (Chilukuri and Bartlett, 1997) binding protein strains toxRS operon pressure sensors P. profundum SS9 2551 (Welch and Bartlett, 1998) ORF3/4 partial EPA fatty acid synthase P. profundum SS9 2551 (Allen et al., 1999) subunit 96 Bartlett

ORF2 ORF3/4 ORF3/4 SS9 ORF5 ORF6 ORF6 ORF3/4

Comparison Vitamin B12 Glycolipid Polyketide ORF3/4 HglC HglD HglC Protein receptor synthase synthase Organism Escherichia Nostoc Amycolatopsis Shewanella Anabaena Anabaena Anabaena coli punctiforme mediterranei SCRC-2738 sp. sp. sp. Accession number P06129 AF01680 AJ223012 U73935 U13677 u13677 U13677 Blast E 1e-12 0 1e-81 e-142 2e-36 3e-90 4e-63 Alignment 14.6 233 43 114 44 98 71

ORF6 ORF6 ORF6 ORF7 ORF7 ORF8 ORF8

Comparison Glycolipid PksC Polyketide Polyketide PksE PleD Response Protein synthase Polyketide synthase synthase? Polyketide regulator synthase synthase? Organism Nostoc Mycobacterium Saccharopolyspora Anabaena Bacillus Synechocystis Aeromonas punctiforme leprae erythraea sp. subtilis sp. jandaei Accession number AF016890 S73013 M63676 U04436 Z99112 D90902 U67070 Blast E 5e-60 6e-49 2e-45 1e-168 4e-86 1e-27 2e-13 Alignment 60 40 38 196 99 27 14

Figure 1. The Shewanella EPA Biosynthetic Genes Similarity of deduced protein sequences from Shewanella SCRC-2738 ORFs and one partial SS9 ORF involved in EPA biosynthesis with additional deduced protein sequences available in GenBank. The position, orientation and size of the ORFs are indicated below. ORFs 2-8 are of Shewanella SCRC-2738 (indicated as ORFS 6, 10, 12, 14, 16, 18 in Genbank U73935; Yazawa, 1996). The significance of the similarities are presented as gapped Blast E values (Altschul et al., 1997) and alignment scores derived from optimized values of RDF2 (Lipman and Pearson, 1985). certain physiological conditions not evaluated in our work. sensors and regulators of gene expression in response to Another possibility is that EPA is not required for changes in temperature, osmolarity, pH and the levels of psychrotolerant or piezotolerant bacterial growth, but as a certain extracellular amino acids (Miller and Mekalanos, nutritional source used by higher organisms with which 1988; Gardel and Mekalanos, 1994). ToxR is an oligomeric EPA producing microoorganisms have established protein whose cytoplasmic domain binds to genes under symbiotic associations. Many of the PUFA producing its control (Dziejman and Mekalanos, 1994; Otteman and microorganisms that have been discovered have been Mekalanos, 1995). ToxS modulates ToxR activity (DiRita isolated from vertebrate or invertebrate sources (Yayanos and Mekalanos, 1991). et al., 1979; Deming et al., 1984; DeLong, 1986; DeLong SS9 ToxR/S pressure sensing appears to depend on and Yayanos, 1986; Yazawa, 1996; Yano et al., 1997). the physical state of the inner membrane. Treatment of Clearly there is a need to better define the roles and SS9 with local anesthetics at concentrations which are regulation of both MUFAs and PUFAs in deep-sea bacteria. known to increase membrane fluidity results in a low- pressure ToxR/S signaling phenotype (high OmpL, low Pressure Regulation of Outer Membrane Proteins in OmpH abundance) even when the cells are grown at high P. profundum pressure (Welch and Bartlett, 1998). We hypothesize that the low pressure mode of ToxR/S regulation represents Another pressure response of strain SS9 is to modulate ToxR/S in its active state, where it is capable of binding to outer membrane protein (OMP) abundance. When cells its cognate regulatory sequences, resulting in either are shifted from atmospheric pressure to higher pressures repression or activation of gene expression. the amount of the outer membrane porin-like protein OmpL High pressure decreases the abundance as well as is lessened and production of the porin-like protein OmpH specific activity of ToxR/S. ToxR (and presumably ToxS) is increased (Bartlett et al., 1989; Chi and Bartlett, 1993; levels decrease with increasing pressure, but even if ToxR/ Welch and Bartlett, 1996; 1998). Although ompL mutants S levels are artificially increased at high pressure by cloning are not impaired in growth at 1 atm and ompH mutants are the genes on a multiple copy plasmid, elevated pressure not altered in growth at elevated pressure, physiological still produces a wild type OMP pattern. In contrast, at experiments with various mutants suggest that OmpH increased temperatures ToxR abundance drops, but OmpL/ provides a larger channel than OmpL (Bartlett and Chi, H levels do not substantially vary, presumably because 1994), a property which could be important in the deep ToxR specific activity increases with temperature. Thus, sea where nutrients are frequently limiting. The ompL and the SS9 ToxR/S system appears well tuned to function as ompH genes are transcriptionally regulated by the inner a barometer (or piezometer) rather than a thermometer. In membrane-localized ToxR and ToxS proteins (Welch and contrast, ToxR/S dependent pressure regulation has not Bartlett, 1998). These proteins were first discovered in been observed in any of the several mesophilic Vibrio spp Vibrio cholerae where they act both as environmental examined (our unpublished results). Microbial Adaptations to the Psychrosphere/Piezosphere 97

Genes Required for Piezoadaptation in P. profundum physiological significance in bacteria adapted to different temperature (and pressure) regimes. As with MUFAs and In addition to the toxRS operon two addition loci have been ToxR/S, RseC represents another membrane-localized uncovered whose products influence OMP abundance. component needed for pressure-adaptation or pressure- These remaining loci are particularly interesting because signal transduction. mutations in them render the cells extremely sensitive to The second gene discovered to be required for piezo- elevated pressure. adaptation of SS9 was isolated by complementation of a The first locus which was found to be required for both pressure-sensitive mutant, EC1002, impaired in the psychro- and piezo-adaptation in strain SS9 was identified expression of a ompH::lacZ transcriptional fusion (Chi and among SS9 transposon mutants which possessed reduced Bartlett, 1993; Bidle and Bartlett, 1999). At high pressure OmpH levels. Transposon cloning and DNA sequence this strain develops into enlarged spherical cells which analysis indicated that insertions in the rseB gene of the divide poorly . The gene responsible for complementation rpoE operon were responsible for these pressure-sensitive of high pressure growth and normal cell morphology was mutants (Chi and Bartlett, 1995). This operon has been found to encode a protein, RecD, which is involved in investigated in several gram-negative bacteria, most homologous DNA recombination. Further study of mutant notably E. coli and Pseudomonas aeruginosa (Deretic et EC1002 confirmed that it possessed phenotypes consistent al., 1994; Lonetto, et al., 1994; Raina et al., 1995; Rouvière with those of a recD mutant, i.e. a deficency in plasmid et al., 1995; Ohman et al., 1996; De Las Peñas et al., 1997; stability, and that this strain harbored a stop codon within Missiakas et al., 1997). In these mesophiles the first gene the recD gene. Two additional recD mutants were in the operon, desginated rpoE in the case of E. coli, constructed by gene disruption and were also found to encodes an alternative sigma factor which activates the possess a pressure-sensitive growth phenotypes. expression of a number of genes in response to damaging The connection between DNA recombination, ompH conditions in the extracytoplasmic compartment. The genes expression and growth and normal cell morphology at high downstream from rpoE are designated rseA, rseB and rseC pressure is still unknown. However, it may be noteworthy in E. coli. The rseA and rseB gene products regulate the that mutants impaired in chromosome partitioning have activity of RpoE in response to the prevailing conditions previously been observed to possess defects in outer within the membranes and periplasmic space. The last membrane proteins (Bahloul et al., 1996). RecD, along with gene in the operon, rseC, is poorly understood. Mutations RecB and RecC are subunits of Exonuclease V, which plays in rseC do not result in a dramatic change in RpoE activity a major role in the homologous recombination pathway of in any of the mesophiles examined and rseC homologs bacteria; reviewed in Myers and Stahl (1994). RecBCD have been found in several bacteria unlinked to rpoE. It is has been proposed to function as a destructive nuclease therefore believed that RseC functions independently of of linear DNA until it encounters the octameric sequence RpoE. Based on the phenotypes of rseC mutants in Chi (χ). At this sequence, RecD is hypothesized to Rhodobacter capsulatus and S. typhimurium it has been dissociate from the complex, while the RecBC enzyme is suggested that RseC proteins function to promote the proposed to convert to a χ-independent recombinogenic assembly of certain membrane-associated protein helicase, to produce single-stranded DNA that serves as complexes (Beck et al., 1997). a substrate for RecA-catalyzed recombination (Dixon et The SS9 rseB insertion mutants were altered in the al., 1994). abundances of numerous outer membrane proteins in Because E. coli recD mutants are hyper- addition to OmpH. The basis for this phenotype was not recombinogenic (Biek and Cohen, 1986), we propose that determined, but could have resulted from excessively high excessive DNA recombination is at the heart of SS9 recD RpoE acitivity resulting from the inactivation of the rseB mutant pressure-sensitivity. If a recD mutant in SS9 exhibits gene. E. coli RpoE controls the expression of the an increased frequency of inter-chromosomal periplasmic protease DegP (Raina et al., 1995; Rouvière recombination and genome concatemerization, then et al., 1995), whose activity could control the turnover of elevated pressure could compromise cell growth by many outer membrane proteins. exacerbating this condition. This could be accomplished High pressure revertants of the SS9 rseB mutants were by further increasing the rate of chromosome readily obtained. These strains were always concomittantly multimerization, or by inhibiting the rate of resolution of restored for low temperature growth ability. Most of these recombined chromosomes. Chromosome multimers would revertants possessed DNA rearrangements at the site of not segregate properly and would thereby block the transposon insertion, further demonstrating the subsequent steps leading to cell division. Thus, impaired importance of the rpoE locus to high pressure and low chromosome segregation could explain the poor growth temperature growth. Complementation analyses indicated and enlarged swollen cells of SS9 recD mutants grown at that rseC is required for piezo- and psychro-adaptation. high pressure. Figure 2 presents a schematic of this model. rseC mutants in E. coli and P. aeruginosa are neither low Another intriguing aspect of SS9 recD is its effect on temperature-sensitive nor increased in piezosensitivity. E. coli cell morphology. Among the earliest observations Thus, while the SS9 rpoE operon is closely related to its of the effects of moderate pressure on bacteria, is that homolog in mesophiles and therefore must function using stressful pressures induce the development of highly essentially the same mechanisms present in other bacteria, filamentous cells (ZoBell and Cobet, 1963; ZoBell, 1970). the functioning of SS9 RseC seems particularly attuned to The introduction of the SS9 recD gene into an E. coli recD low temperature and high pressure conditions. Clarification mutant prevented cell filamentation at elevated pressures of these distinctions could provide new insight into the (Bidle and Bartlett, 1999). This effect was recessive to wild function of the rpoE operon gene products and their type E. coli recD. 98 Bartlett

elevated levels in deep-sea versus shallow-water fishes (Gillett et al., 1997). Furthermore, these investigators have found that TMAO reduces lactate dehydrogenase Km at high pressure. One possibility is that certain osmolytes which are excluded from the hydration layers of proteins (recD + ) (recD - ) could be used by deep-sea organisms to help counterbalance the protein hydrating influence of high pressure.

Concluding Remarks

There are opportunities for major discoveries to be made in deep-sea microbiology across a spectrum of levels of study extending from issues of biodiversity to the identification of genes required for piezoadaptation to membrane and protein biophysical studies at high pressure. With regard to the main focus of this review, the Figure 2. Schematic of the Possible Role of RecD in Preventing identification and elucidation of genes required for sensing Interchromosomal Recombination in SS9 at Elevated Pressure and adapting to changes in hydrostatic pressure is still in its infancy. At this juncture it appears that genes which control membrane structure and DNA recombination are Protein Adaptation particularly important. But there are many possible pressure points in a bacterial cell, and many genetic modifications Studies of pressure effects on proteins from shallow and that must undoubtedly occur to enable life in the deep-sea animals indicate that deep-sea fishes and piezosphere. invertebrates possess proteins with structural and functional adaptations to high pressure (Somero, 1990; Acknowledgements 1991; 1992). Unfortunately similar comparisons have not been undertaken for proteins from shallow and deep-sea D.H.B. gratefully acknowledges support from the National Science bacteria, and no definitive data exists for the types of amino Foundation. acid changes favored at high pressure. However, one References comparison has been made of homologous proteins from related bacteria differing in pressure adaptation. This was Allen, E.E, Facciotti, D., and Bartlett, D.H. 1999. Monounsaturated but not performed for the oligomeric single-stranded DNA-binding polyunsaturated fatty acids are required for growth at high pressure and protein (SSB) from four closely related marine shewanellas low temperature in the deep-sea bacterium Photobacterium profundum strain SS9. Appl. Environ. Microbiol. 65: 1710-1720. whose pressure optima ranged from atmospheric pressure Altschul, S.F., Madden, T. L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, to 69 MPa (Chilukuri and Bartlett, 1997). The Shewanella W., and Lipman, D.J. 1997. Gapped BLAST amd PSI-BLAST: a new SSBs could be divided into conserved amino- and carboxy- generation of protein database search programs. Nucleic Acids Res. 25: terminal regions and a highly variable central region. 3389-3402. Bahloul, A., Meury, J., Kern, R., Garwood, J., Guha, S., and Kohiyama, M. Analysis of the amino acid composition of the Shewanella 1996. 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